The present invention encompasses radiolabeled peptide analogs of vasoactive intestinal peptide (VIP) labeled with a radionuclide useful for imaging target sites within mammalian living systems. The invention particularly provides radiolabeled VIP derivatives that bind selectively to the VIP receptor on target cells. Specifically, the invention relates to the radiolabeling of VIP-receptor specific agents and their subsequent use for radiodiagnostic and radiotherapeutic purposes. The invention encompasses methods for radiolabeling these peptides with radionuclides and the use of these peptides as scintigraphic imaging agents. The radiolabeled VIP derivatives of the present invention exhibit pharmacological activity and therefore are useful as either imaging agent for visualization of VIP-receptor positive tumors and metastases, as a radiodiagnostic agent or as a radio-therapeutic agent for the treatment of such tumors in vivo by specifically targeting the cytotoxic radionuclide selectively to the tumor site in mammalian living systems.
Vasoactive intestinal peptide is a 28-amino acid neuropeptide, which was first isolated from the porcine intestine (Said and Mutt, 1970). It bears extensive homology to secretin, PHI and glucagon. The amino acid sequence for VIP is:
His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (SEQ ID NO: 1)
VIP is known to exhibit a wide variety of biological activities such as the autocrine, endocrine and paracrine functions in living organisms (Said, 1984). In the gastrointestinal tract, it has been known to stimulate pancreatic and biliary secretions, hepatic glycogenesis as well as the secretion of insulin and glucagon (Kerrins and Said, 1972; Domschke et al., 1977). In the nervous system it acts as a neurotransmitter and neuromodulator, regulating the release and secretion of several key hormones (Said, 1984). In recent years, attention has been focussed on the function of VIP in certain areas of the CNS as well its role in the progression and control of neoplastic disease (Reubi, 1995).
The importance of peptide growth factors and regulatory hormones in the etiology and pathogenesis in several carcinomas has long been recognized. Data from epidemiological and endocrinological studies suggest that neuropeptides like VIP which are responsible for the normal growth of tissues like the pancreas can also cause conditions for their neoplastic transformation (Sporn et al., 1980). Several lines of evidence indicate that VIP acts as a growth factor and plays a dominant autocrine and paracrine role in the sustained proliferation of cancer cells (Said, 1984). The stimulatory effect of VIP on tumor growth can be mediated directly by its receptors on cell membranes or indirectly by potentiation of the activities of other growth factors in tumor cells (Scholar E. M. Cancer 67(6): 1561-1569, 1991). The synergistic effect of VIP and related pituitary adenylate cyclase activating polypeptide (PACAP) in glioblastomas is an illustration to the above fact (Moody, T. W., et al. Peptides 17(3), 545-555, 1996).
The multiple physiological and pharmacological activities of VIP are mediated by high affinity G-protein coupled transmembrane receptors on target cells (Reubi, 1995). VIP receptors are coupled to cellular effector systems via adenylyl cyclase activity (Xia et al., 1996). The VIP receptor, found to be highly over-expressed in neoplastic cells, is thought to be one of the biomarkers in human cancers (Reubi, 1995). High affinity VIP receptors have been localized and characterized in neoplastic cells of most breast carcinomas, breast and prostate cancer metastases, ovarian, colonic and pancreatic adenocarcinomas, endometrial and squamous cell carcinomas, non small cell lung cancer, lymphomas, glioblastomas, astrocytomas, meningiomas and tumors of mesenchymal origin. Amongst, neuroendocrine tumors all differentiated and non-diffemtiated gastroenteropancreatic tumors, pheochromocytomas, small-cell lung cancers, neuroblastomas, pituitary adenomas as well tumors associated with hypersecretory states like Verner-Morrison syndrome were found to overexpress receptors for vasoactive intestinal peptide (Reubi, 1995, 1996, 1999; Tang et al., 1997aandb; Moody et al., 1998aandb; Waschek et al., 1995; Oka et al., 1998)). These findings suggest that new approaches for the diagnosis and treatment of these cancers may be based on functional manipulation of VIP activity, using synthetic peptide analogs of the same.
Historically, the somatostatin analog 111In-DTPA-[D-Phe1]-octreotide is the only radiopeptide, which has obtained regulatory approval in USA and Europe (Lamberts et al., 1995). Radiolabeled VIP has been shown to visualize a majority of gastropancreatic adenocarcinomas, neuroendocrine tumors, as well as insulinomas (which are often missed by radiolabeled octreotide) (Behr et al., 1999). VIP-receptor scinitigraphy offers certain advantages over radioimaging involving somatostatin receptors. The presence of high affinity receptors for VIP have been demonstrated in a larger number of human tumors, relative to the somatostatin receptors. Secondly, the density of VIP receptors on tumors has been found to be greater than somatostatin (Behr et al., 1999). Therefore, the VIP-receptor scan is more sensitive and convenient in localizing tumors and their metastatic spread as compared to somatostatin. The applications of this technique are manifold. It has been used for the sensitive detection of VIP-receptor positive tumors. This includes primary carcinoids, cancers of the gastrointestinal tract as well as distant metastases (Reubi, 1995, 1996). It can also be used to target cytotoxic radionuclides specifically to the tumor site. It predicts the VIP-receptor status of the patient and thereby the response of the patient towards radiotherapy by radiolabeled VIP analogs. Lastly, such radiolabeled peptides have been successfully used in radioguided surgery (Lamberts et al., 1995).
123I-VIP, 125I-VIP and their derivatives have been extensively used for imaging pancreatic adenocarcinomas, endocrine tumors of the gastrointestinal origin, mesenchymal tumors as well secondary tumor metastatic sites, in patients (Jiang et al., 1997; Virgolini et al., 1996, 1998; Raderer et al., 1998 ; Moody et al., 1998; Kurtaran et al., 1997; Pallella et al., 1999). Radioiodinated VIP and its derivatives have been also used to assess the binding affnty of peptides for VIP-receptors on tumor cells in vitro. The biodistribution, safety and absorbed dose of the aforesaid radioiodinated peptide derivatives have also been studied earlier (Virgolini et al., 1995).
U.S. Pat. No. 5,849,261, granted to Dean et al., on Dec. 15, 1998 describes the applications of radiolabeled vasoactive intestinal peptide (VIP) for diagnosis and therapy. In particular, this U.S. Patent discloses a method for preparing a radiopharmaceutical agent, comprising native vasoactive intestinal (VIP) peptide attached to a radionuclide like technetium or rhenium via a chelating moiety. The radiopharmaceutical when labeled with technetium or rhenium via a chelating moiety has a VIP binding affinity which is not less than about one tenth the affinity of radioiodinated native VIP for the receptor.
However, there is still a need for improved synthetic analogs of VIP as radiopharmaceuticals, which are easy to generate and are capable of being employed with higher sensitivity and specificity in terms of their radioimaging and radiodiagnostic properties.
This invention describes the preparation and use of peptide analogs of VIP having constrained amino acids. The design of conformationally constrained bioactive peptide derivatives has been one of the widely used approaches for the development of.peptide-based therapeutic agents. Non-standard amino acids with strong conformational preferences may be used to direct the course of polypeptide chain folding, by imposing local stereochemical constraints, in de novo approaches to peptide design. The conformational characteristics of xcex1,xcex1-dialkylated amino acids have been well studied. The incorporation of these amino acids restricts the rotation of xcfx86, xcexa8 angles, within the molecule, thereby stabilizing a desired peptide conformation.
The prototypic member of xcex1,xcex1-dialkylated aminoacids, xcex1-amino-isobutyric acid (Aib) or xcex1,xcex1-dimethylglycine has been shown to induce xcex2-turn or helical conformation when incorporated in a peptide sequence (Prasad and Balaram, 1984, Karle and Balaram, 1990). The conformational properties of the higher homologs of xcex1,xcex1-dialkylated amino acids such as di-ethylglycine (Deg), di-n-propylglycine (Dpg), di-n-butylglycine (Dbg) as well as the cyclic side chain analogs of xcex1,xcex1-dialkylated amino acids such as 1-aminocyclopentane carboxylic acid (Ac5c), 1 -aminocyclohexane carboxylic acid (Ac6c), 1-aminocycloheptane carboxylic acid (Ac7c) and 1-aminocyclooctane carboxylic acid (Ac8c) have also been shown to induce folded conformation (Prasad et al., 1995 ; Karle et al., 1995). xcex1,xcex1-dialkylated amino acids have been used in the design of highly potent chemotactic peptide analogs (Prasad et al., 1996). The present invention incorporates the conformational properties of such xcex1,xcex1-dialkylated amino acids for the design of biologically active peptide derivatives, taking VIP as the model system under consideration.
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Throughout the specification and claims the following abbreviations are used:
Aib: xcex1-Aminoisobutyric acid
Deg: xcex1,xcex1-Diethylglycine
Ac5c: 1-Amino Cyclopentane Carboxylic acid
BOP: Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexofluorophosphate
PyBOP: Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexofluorophospate
HBTU: O-Benzotriazole-N,N,Nxe2x80x2,Nxe2x80x2-tetramethyl-uronium-hexofluoro-phosphate
TBTU: 2-(1H-Benzotriazole-1-yl)-1, 1, 3, 3-tetramethyluronium tetrafluoroborate
HOBt: 1-Hydroxy Benzotriazole
DCC: Dicyclohexyl carbodiimide
DIPCDI: Diisopropyl carbodiimide
DIEA: Diisopropyl ethylamine
DMF: Dimethyl formamide
DCM: Dichloromethane
NMP: N-Methyl-2-pyrrolidinone
TFA: trifluoroacetic acid
Throughout the specification and claims the amino acid residues are designated by their standard abbreviations. Amino acids denote L-configuration unless indicated by D or DL appearing before the symbol and separated from it by a hypen.
The present invention encompasses radiolabeled peptide analogs of vasoactive intestinal peptide (VIP) labeled with a radionuclide useful for imaging target sites (e.g. use as a scintigraphic imaging agent), for use in radiodiagnostics, and radiotherapy within mammalian living systems. The invention particularly provides radio labeled VIP derivatives that bind selectively to the VIP receptor on target cells. Specifically, the invention relates to the radiolabeling of VIP receptor specific agents and their subsequent use for radiodiagnostic and radiotherapeutic purposes. The invention encompasses methods for radiolabeling these peptides with radionuclides and the use of these peptides as scintigraphic imaging agents. The present invention also encompasses the use of these radiolabeled peptides as anti-neoplastic agents for specific radiotherapy in cancer. A further object of the invention is the use of certain novel VIP analogs to determine the binding affinities of these peptides for their cognate receptors on cancer cells.